Chimeric antibodies with part new world primate binding regions

ABSTRACT

The present invention provides a chimeric antibody polypeptide comprising an antigen binding site, wherein the antigen binding site comprises a human variable domain having at least one New World Primate CDR.

FIELD OF THE INVENTION

The present invention relates to engineered antibody polypeptides. Moreparticularly, the present invention provides antibody polypeptidescomprising an antigen binding site, wherein the antigen binding sitecomprises a human variable domain having at least one New World PrimateCDR. In particular the present invention relates to antibodypolypeptides directed against TNF-α.

BACKGROUND OF THE INVENTION

As the name implies, Tumor Necrosis Factor-α (TNF-α) was originallydescribed as a molecule having anti-tumor properties, but the moleculewas subsequently found to play key roles in other processes, including aprominent role in mediating inflammation and autoimmune disorders. TNF-αis a key proinflammatory cytokine in inflammatory conditions including,for example, rheumatoid arthritis (RA), Crohn's disease, ulcerativecolitis and other bowel disorders, psoriasis, toxic shock, graft versushost disease and multiple sclerosis. The pro-inflammatory actions ofTNF-α result in tissue injury, such as inducing procoagulant activity onvascular endothelial cells (Pober, et al., 1986, J. Immunol.136:1680-1687), increasing the adherence of neutrophils and lymphocytes(Pober, et al., 1987, J. Immunol. 138:3319-3324), and stimulating therelease of platelet activating factor from macrophages, neutrophils andvascular endothelial cells (Camussi, et al., 1987, J. Exp. Med.166:1390-1404). TNF-α is synthesized as a 26 kD transmembrane precursorprotein with an intracellular tail that is cleaved by a TNF-α-convertingmetalloproteinase enzyme and then secreted as a 17 kD soluble protein.The active form consists of a homotrimer of the 17 kD monomers whichinteracts with two different cell surface receptors, p55 TNFR1 and p75TNFR2. There is also evidence that the cell surface bound precursor formof TNF-α can mediate some biological effects of the factor. Most cellsexpress both p55 and p75 receptors which mediate different biologicalfunctions of the ligand. The p75 receptor is implicated in triggeringlymphocyte proliferation, and the p55 receptor is implicated inTNF-mediated cytotoxicity, apoptosis, antiviral activity, fibroblastproliferation and NT-κB activation (see Locksley et al., 2001, Cell 104:487-501). The TNF receptors are members of a family of membrane proteinsincluding the NGF receptor, Fas antigen, CD27, CD30, CD40, Ox40 and thereceptor for the lymphotoxin α/β heterodimer. Binding of receptor by thehomotrimer induces aggregation of receptors into small clusters of twoor three molecules of either p55 or p75. TNF-α is produced primarily byactivated macrophages and T lymphocytes, but also by neutrophils,endothelial cells, keratinocytes and fibroblasts during acuteinflammatory reactions. TNF-α is at the apex of the cascade ofpro-inflammatory cytokines (Reviewed in Feldmann & Maini, 2001, Ann.Rev. Immunol. 19: 163-196). This cytokine induces the expression orrelease of additional proinflammatory cytokines, particularly IL-1 undIL-6 (see, for example, Rutgeerts et al., 2004, Gastroenterology 126:1593-1610). Inhibition of TNF-α inhibits the production of inflammatorycytokines including IL-1, IL-6, IL-8 and GM-CSF (Brennan et al., 1989,Lancet 2: 244-247). Because of its role in inflammation, TNF-α hasemerged as an important inhibition target in efforts to reduce thesymptoms of inflammatory disorders. Various approaches to inhibition ofTNF-α for the clinical treatment of disease have been pursued, includingparticularly the use of soluble TNF-α receptors and antibodies specificfor TNF-α. Commercial products approved for clinical use include, forexample, the antibody products Remicade™ (infliximab; Centocor, Malvern,Pa.; a chimeric monoclonal IgG antibody bearing human IgG1 constant andmouse variable regions), Humira™ (adalimumab or D2E7; AbbottLaboratories, described in U.S. Pat. No. 6,090,382) and the solublereceptor product Enbrel™ (etancrcept, a soluble p75 TNFR2 Fc fusionprotein; Immunex). The role of TNF-α in inflammatory arthritis isreviewed in, for example, Li & Schwartz, 2003, Springer Semin.Immunopathol. 25: 19-33. In RA, TNF-α is highly expressed in inflamedsynovium, particularly at the cartilage-pannus junction (DiGiovine etal., 1988, Ann. Rheum. Dis. 47: 768-772; Firestein et al., 1990, J.Immunol. 144: 3347-3353; and Saxne et al., 1988, Arthritis Rheum. 31:1041-1045). In addition to evidence that TNF-α increases the levels ofinflammatory cytokines IL-1, IL-6, IL-8 and GM-CSF, TNF-α can alonetrigger joint inflammation and proliferation of fibroblast-likesynoviocytes (Gitter et al., 1989, Immunology 66: 196-200), inducecollagenase, thereby triggering cartilage destruction (Dayer et al.,1985, J. Exp. Med. 162: 2163-2168; Dayer et al., 1986, J. Clin. Invest.77: 645-648), inhibit proteoglycan synthesis by articular chondrocytes(Saklatvala, 1986, Nature 322: 547-548; Saklatvala et al., 1985, J. Exp.Med. 162; 1208-1222) and can stimulate osteoclastogenesis and boneresorption (Abu-Amer et al., 2000, J. Biol. Chem., 275: 27307-27310;Bertolini et al., 1986, Nature 319: 516-518). TNF-α induces increasedrelease of CD14+ monocytes by the bone marrow. Such monocytes caninfiltrate joints and amplify the inflammatory response via the RANK(Receptor Activator of NF-κB)-RANKL signaling pathway, giving rise toosteoclast formation during arthritic inflammation (reviewed inAnandarajah & Richlin, 2004, Curr. Opin. Rheumatol. 16: 338-343). TNF-αis an acute phase protein which increases vascular permeability throughits induction of IL-8, thereby recruiting macrophage and neutrophils toa site of infection. Once present, activated macrophages continue toproduce TNF-α, thereby maintaining and amplifying the inflammatoryresponse. Titration of TNF-α by the soluble receptor constructetanercept has proved effective for the treatment of RA, but not fortreatment of Crohn's disease. In contrast, the antibody TNF-α antagonistinfliximab is effective to treat both RA and Crohn's disease. Thus, themere neutralization of soluble TNF-α is not the only mechanism involvedin anti-TNF-based therapeutic efficacy. Rather, the blockade of otherpro-inflammatory signals or molecules that are induced by TNF-α alsoplays a role (Rutgeerts et al., supra). For example, the administrationof infliximab apparently decreases the expression of adhesion molecules,resulting in a decreased infiltration of neutrophils to sites ofinflammation. Also, infliximab therapy results in the disappearance ofinflammatory cells from previously inflamed bowel mucosa in Crohn'sdisease. This disappearance of activated T cells in the lamina propriais mediated by apoptosis of cells carrying membrane-bound TNF-αfollowing activation of caspases 8,9 and then 3 in a Fas dependentmanner (see Lugering et al., 2001, Gastroenterology 121: 1145-1157).Thus, membrane- or receptor-bound TNF-α is an important target foranti-TNF-α therapeutic approaches. Others have shown that infliximabbinds to activated peripheral blood cells and lamina propria cells andinduces apoptosis through activation of caspase 3 (sec Van den Brando etal., 2003, Gastroenterology 124: 1774-1785). Intracellularly, thebinding of trimeric TNF-α to its receptor triggers a cascade ofsignaling events, including displacement of inhibitory molecules such asSODD (silencer of death domains) and binding of the adaptor factorsFADD, TRADD, TRAF2, c-IAP, RAIDD and TRIP plus the kinase RIP1 andcertain caspases (reviewed by Chen & Goeddel, 2002, Science 296:1634-1635, and by Muzio & Saccani in :Methods in Molecular Medicine:Tumor Necrosis Factor, Methods and Protocols,” Corti and Ghezzi, eds.(Humana Press, New Jersey; 2004), pp. 81 -99). The assembled signallingcomplex can activate either a cell survival pathway, through NF-κBactivation and subsequent downstream gene activation, or an apoptoticpathway through caspase activation. Similar extracellular downstreamcytokine cascades and intracellular signal transduction pathways can beinduced by TNF-α in other diseases. Thus, for other diseases ordisorders in which the TNF-α molecule contributes to the pathology,inhibition of TNF-α presents an approach to treatment. Angiogenesisplays an important role in the active proliferation of inflammatorysynovial tissue. RA synovial tissue, which is highly vascularized,invades the periarticular cartilage and bone tissue and leads to jointdestruction. Vascular endothelial growth factor (VEGF) is the mostpotent angiogenic cytokine known. VEGF is a secreted, heparin-binding,homodimeric glycoprotein existing in several alternate forms due toalternative splicing of its primly transcript (Leung et al., 1989,Science 246: 1306-1309). VEGF is also known as vascular permeabilityfactor (VPF) due to its ability to induce vascular leakage, a processimportant in inflammation. The identification of VEGF in synovialtissues of RA patients highlighted the potential role of VEGF in thepathology of RA (Fava et al., 1994, J. Exp. Med. 180: 341-346; Nagashimaet al., 1995, J. Rheumatol. 22: 1624-1630). A role for VEGF in thepathology of RA was solidified following studies in which anti-VEGFantibodies were administered in the murine collagen-induced arthritis(CIA) model. In these studies, VEGF expression in the joints increasedupon induction of the disease, and the administration of anti-VEGFantisera blocked the development of arthritic disease and amelioratedestablished disease (Sone et al., 2001, Biochem. Biophys. Res. Comm.281: 562-568; Lu et al., 2000, J. Immunol. 164: 5922-5927).

Antibody Polypeptides

Antibodies are highly specific for their binding targets and althoughthey are derived from nature's own defence mechanisms, antibodies faceseveral challenges when applied to the treatment of disease inhumanpatients. Conventional antibodies are large multi-subunit proteinmolecules comprising at least four polypeptide chains. For example,human IgG has two heavy chains and two light chains that are disulfidebonded to form the functional antibody. The size of a conventional IgGis about 150 kD. Because of their relatively large size, completeantibodies (e.g., IgG, IgA, IgM, etc.) are limited in their therapeuticusefulness due to problems in, for example, tissue penetration.Considerable efforts have focused on identifying and producing smallerantibody fragments that retain antigen binding function and solubility.The heavy and light polypeptide chains of antibodies comprise variable(V) regions that directly participate in antigen interactions, andconstant (C) regions that provide structural support and function innon-antigen-specific interactions with immune effectors. The antigenbinding domain of a conventional antibody is comprised of two separatedomains: a heavy chain variable domain (VH) and a light chain variabledomain (VL: which can be either Vκ or Vλ). The antigen binding siteitself is formed by six polypeptide loops: three from the VH domain (H1,H2 and H3) and three from the VL domain (L1, L2 and L3). In vivo, adiverse primary repertoire of V genes that encode the VH and VL domainsis produced by the combinatorial rearrangement of gene segments. Cregions include the light chain C regions (referred to as CL regions)and the heavy chain C regions (referred to as CH1, CH2 and CH3 regions).A number of smaller antigen binding fragments of naturally occurringantibodies have been identified following protease digestion. Theseinclude, for example, the “Fab fragment” (VL-CL-CH1-VH), “Fab′ fragment”(a Fab with the heavy chain hinge region) and “F(ab′)2 fragment” (adimer of Fab′ fragments joined by the heavy chain hinge region).Recombinant methods have been used to generate even smallerantigen-binding fragments, referred to as “single chain Fv” (variablefragment) or “scFv,” consisting of VL and VH joined by a syntheticpeptide linker.

Single Domain Antibodies

While the antigen binding unit of a naturally-occurring antibody (e.g.,in humans and most other mammals) is generally known to be comprised ofa pair of V regions (VL/VH), camelid species express a large proportionof fully functional, highly specific antibodies that are devoid of lightchain sequences. The camelid heavy chain antibodies are found ashomodimers of a single heavy chain, dimerized via their constantregions. The variable domains of these camelid heavy chain antibodiesare referred to as VHH domains and retain the ability, when isolated asfragments of the VH chain, to bind antigen with high specificity(Hamers-Casterman et al., 1993, Nature 363: 446-448; Gahroudi et al.,1997, FEBS Lett. 414: 521 -526). Antigen binding single VH domains havealso been identified from, for example, a library of murine VH genesamplified from genomic DNA from the spleens of immunized mice andexpressed in E. coli (Ward et al, 1989, Nature 341: 544-546). Ward etal. named the isolated single VH domains “dAbs,” for “domainantibodies”. The term “dAb” will refer herein to a single immunoglobulinvariable domain (VH, VHH or VL) polypeptide that specifically bindsantigen. A “dAb” binds antigen independently of other V domains;however, as the term is used herein, a “dAb” can be present in a homo-or heteromultimer with other VH or VL domains where the other domainsare not required for antigen binding by the dAb, i.e., where the dAbbinds antigen independently of the additional VH, VHH or VL domains.Single immunoglobulin variable domains, for example, VHH, are thesmallest antigen-binding antibody unit known. For use in therapy, humanantibodies are preferred, primarily because they are not as likely toprovoke an immune response when administered to a patient. Isolatednon-camelid VH domains tend to be relatively insoluble and are oftenpoorly expressed. Comparisons of camelid VHH with the VH domains ofhuman antibodies reveals several key differences in the frameworkregions of the camelid VHH domain corresponding to the VR/VL interfaceof the human VH domains. Mutation of these residues of human VH3 to moreclosely resemble the VHH sequence (specifically Gly 44 Glu, Leu 45 Argand Trp 47 Gly) has been performed to produce “camelized” human VHdomains (Davies & Riechmann, 1994, FEBS Lett. 339: 285-290) in anattempt to yield improved expression and solubility. Variable domainamino acid numbering used herein is consistent with the Kabat numberingconvention (Kabat et al., 1991, Sequences of Immunological Merest, 5thed. U.S. Dept. Health & Human Services, Washington, D.C.).

WO 03/035694 (Muyldermans) reports that a Trp 103 Arg mutation improvesthe solubility of non-camelid VH domains, Davies & Riechmann, (1995,Biotechnology N.Y. 13: 475-479) also report production of aphage-displayed repertoire of camelized human VH domains and selectionof clones that bind hapten with affinities in the range of 100-400 nM,but clones selected for binding to protein antigen had weakeraffinities. The antigen binding domain of an antibody comprises twoseparate regions: a heavy chain variable domain (VH) and a light chainvariable domain (VL: which can be either Vκ or Vλ). The antigen bindingsite itself is formed by six polypeptide loops: three from VH domain(H1, H2 and H3) and three from VL domain (L1, L2 and L3). A diverseprimary repertoire of V genes that encode the VH and VL domains isproduced by the combinatorial rearrangement of gene segments. The VHgene is produced by the recombination of three gene segments, VH, D andJH. In humans, there are approximately 51 functional VH segments (Cookand Tomlinson, 1995, Immunol. Today, 16: 237), 25 functional D segments(Corbett et al., 1997 J. Mol. Biol., 268: 69) and 6 functional JHsegments (Ravetch et al., 1981, Cell, 27: 583-591), depending on thehaplotype. The VH segment encodes the region of the polypeptide chainwhich forms the first and second antigen binding loops of the VH domain(H1 and H2), whilst the VH, D and JH segments combine to form the thirdantigen binding loop of the VH domain (H3). The VL gene is produced bythe recombination of only two gene segments, VL and JL. In humans, thereare approximately 40 functional Vκ segments (Schable and Zachau (1993)Biol. Chem. Hoppe Scyler, 374: 1001-1022), 31 functional Vλ segments(Williams et al., 1996, J. Mol. Biol., 264: 220-232; Kawasaki et al.,1997, Genome Res., 7: 250-261), 5 functional Jκ segments (Hieter et al.,1982, J. Biol. Chef., 257: 1516-1522) and 4 functional Jλ segments(Vasicek and Leder, 1990, J. Exp. Med., 172: 609-620), depending on thehaplotype. The VL segment encodes the region of the polypeptide chainwhich forms the first and second antigen binding loops of the VL domain(L1 and L2), whilst the VL and JL segments combine to form the thirdantigen binding loop of the VL domain (L3).

Antibodies selected from this primary repertoire are believed to besufficiently diverse to bind almost all antigens with at least moderateaffinity. High affinity antibodies are produced by “affinity maturation”of the rearranged genes, in which point mutations are generated andselected by the immune system on the basis of improved binding. Analysisof the structures and sequences of antibodies has shown that five of thesix antigen binding loops (H1, H2, L1, L2, L3) possess a limited numberof main-chain conformations or canonical structures (Chothia and Lesk,1987, Mol. Biol., 196; 901-917; Chothia al., 1989, Nature, 342:877-883). The main-chain conformations are determined by (i) the lengthof the antigen binding loop, and (ii) particular residues, or types ofresidue, at certain key position in the antigen binding loop and theantibody framework. Analysis of the loop lengths and key residues hasenabled us to predict the main-chain conformations of H1, H2, L1, L2 andL3 encoded by the majority of human antibody sequences (Chothia et al.,1992, J. Mol. Biol., 227: 799-817; Tomlinson et al., 1995, EMBO J., 14;4628-4638; Williams et al., 1996, J. Mol. Biol., 264: 220-232). Althoughthe H3 region is much more diverse in terms of sequence, length andstructure (due to the use of D segments), it also forms a limited numberof main-chain conformations for short loop lengths which depend on thelength and the presence of particular residues, or types of residue, atkey positions in the loop and the antibody framework (Martin et al.,1996, J. Mol. Biol, 263: 800-815; Shirai et al., 1996, FEBS Letters,399: 1-8).

Bispecific antibodies comprising complementary pairs of VH and VLregions are known in the art. These bispecific antibodies must comprisetwo pairs of VH and VLs, each VH/VL pair binding to a single antigen orepitope. Methods described involve hybrid hybridomas (Milstein & Cuello,Nature, 1983, 305:537-40), minibodies (Hu et al., 1996, Cancer Res 3056:3055-3061), diabodies (Holliger et al., 1993, Proc. Natl. Acad. Sci.USA 90, 6444-6448; WO 94/13804), chelating recombinant antibodies(CRAbs; Neri et al., 1995, J. Mol. Biol. 246, 367-373), biscFv (e.g.Atwell et al., 1996, Mol. Immunol. 33, 1301-1312), “knobs in holes”stabilized antibodies (Carter et al., 1997, Protein Sci. 6, 781-788). Ineach case, each antibody species comprises two antigen-binding sites,each fashioned by a complementary pair of VH and VL domains. Eachantibody is thereby able to bind to two different antigens or epitopesat the same time, with the binding to EACH antigen or epitope mediatedby a VH and its complementary VL domain. Each of these techniquespresents its particular disadvantages; for instance in the case ofhybrid hybridomas, inactive VH/VL pairs can greatly reduce the fractionof bispecific IgG. Furthermore, most bispecific approaches rely on theassociation of the different VH/VL pairs or the association of VH and VLchains to recreate the two different VH/VL binding sites. It istherefore impossible to control the ratio of binding sites to eachantigen or epitope in the assembled molecule and thus many of theassembled molecules will bind to one antigen or epitope but not theother. In some cases it has been possible to engineer the heavy or lightchains at the sub-unit interfaces (Carter et al., 1997) in order toimprove the number of molecules which have binding sites to bothantigens or epitopes, but this never results in all molecules havingbinding to both antigens or epitopes. There is some evidence that twodifferent antibody binding specificities might be incorporated into thesame binding site, but these generally represent two or morespecificities that correspond to structurally related antigens orepitopes or to antibodies that are broadly cross-reactive. For example,cross-reactive antibodies have been so described, usually where the twoantigens are related in sequence and structure, such as hen egg whitelysozyme and turkey lysozyme (McCafferty et al., WO 92/01047) or to freehapten and to hapten conjugated to carrier (Griffiths et al., 1994, EMBOJ 13:14 3245-60). In a further example, WO 02/02773 (AbbottLaboratories) describes antibody molecules with “dual specificity”. Theantibody molecules referred to are antibodies raised or selected againstmultiple antigens, such that their specificity spans more than a singleantigen. Each complementary VH/VL pair in the antibodies of WO 02/02773specifies a single binding specificity for two or more structurallyrelated antigens; the VH and VL domains in such complementary pairs donot each possess a separate specificity. The antibodies thus have abroad single specificity which encompasses two antigens, which arestructurally related. Furthermore natural autoantibodies have beendescribed that are polyreactive (Casali & Nolkins, 1989, Ann. Rev.Immunol. 7, 515-531), reacting with at least two (usually more)different antigens or epitopes mat are not structurally related. It hasalso been shown that selections of random peptide repertoires usingphage display technology on a monoclonal antibody will identify a rangeof peptide sequences that fit the antigen binding site. Some of thesequences are highly related, fitting a consensus sequence, whereasothers are very different and have been termed mimotopes (Lane &Stephen, 1993, Current Opinion in Immunology, 5,268-271). It istherefore clear that a natural four-chain antibody, comprisingassociated and complementary VH and VL domains, has the potential tobind to many different antigens from a large universe of known antigens.It is less clear how to create a binding site to two given antigens inthe same antibody, particularly those which are not necessarilystructurally related. Protein engineering methods have been suggestedthat may have a bearing on this. For example, it has also been proposedthat a catalytic antibody could be created with a binding activity to ametal ion through one variable domain, and to a hapten (substrate)through contacts with the metal ion and a complementary variable domain(Barbae et al, 1993, Proc. Natl. Acad. Sci USA 90,6385-6389). However inthis case, the binding and catalysis of the substrate (first antigen) isproposed to require the binding of the metal ion (second antigen). Thusthe binding to the VH/VL pairing relates to a single but multi componentantigen. Methods have been described for the creation of bispecificantibodies from camel antibody heavy chain single domains in whichbinding contacts for one antigen are created in one variable domain, andfor a second antigen in a second variable domain. However the variabledomains were not complementary. Thus a first heavy chain variable domainis selected against a first antigen, and a second heavy chain variabledomain against a second antigen, and then both domains are linkedtogether on the same chain to give a bispecific antibody fragment(Conrath et al, J. Biol. Chem. 270, 27589-27594). However the camelheavy chain single domains are unusual in that they are derived fromnatural camel antibodies which have no light chains, and indeed theheavy chain single domains are unable to associate with camel lightchains to form complementary VH and VL pairs. Single heavy chainvariable domains have also been described, derived from naturalantibodies which are normally associated with light chains (frommonoclonal antibodies or from repertoires of domains; see EP-A-0368684).These heavy chain variable domains have been shown to interactspecifically with one or more related antigens but have not beencombined with other heavy or light chain variable domains to create aligand with specificity for two or more different antigens. Furthermore,these single domains have been shown to have a very short in vivahalf-life. Therefore, such domains are of limited therapeutic value. Ithas been suggested to make bispecific antibody fragments by linkingheavy chain variable domains of different specificity together (asdescribed above). The disadvantage with this approach is that isolatedantibody variable domains may have a hydrophobic interface that normallymakes interactions with the light chain and is exposed to solvent andmay be “sticky” allowing the single domain to bind to hydrophobicsurfaces. Furthermore, in the absence of a partner light chain, thecombination of two or more different heavy chain variable domains andtheir association, possibly via their hydrophobic interfaces, mayprevent them from binding to one or both of the ligands they are able tobind in isolation. Moreover, in this case the heavy chain variabledomains would not be associated with complementary light chain variabledomains and thus may be less stable and readily unfold (Worn &Pluckthun, 1998, Biochemistry 37:13120-7).

Human/mouse chimeric antibodies have been created in which antibodyvariable region sequences from the mouse genome are combined withantibody constant region sequences from the human genome. The chimericantibodies exhibit the binding characteristics of the parental mouseantibody, and the effector functions associated with the human constantregion. The antibodies are produced by expression in a host cell,including for example Chinese Hamster Ovary (CHO). NS0 myeloma cells,COS cells and SP2 cells.

Such chimeric antibodies have been used inhuman therapy, howeverantibodies to these chimeric antibodies have been produced by the humanrecipient. Such anti-chimeric antibodies are detrimental to continuedtherapy with chimeric antibodies.

It has been suggested that human monoclonal antibodies are expected tobe an improvement over mouse monoclonal antibodies for in vivo humantherapy. From work done with antibodies from Old World primates (rhesusmonkeys and chimpanzees) it has been postulated mat these non-humanprimate antibodies will be tolerated in humans because they arestructurally similar to human antibodies (Ehrlich PH et al., 1988, Humanand primate monoclonal antibodies for in vivo therapy. Clin Chem. 34:9pg 1681-1688). Furthermore, because human antibodies are non-immunogenicin Rhesus monkeys (Ehrlich et al., 1987, Hybridoma; 6:151-60), it islikely that the converse is also applicable and primate antibodies willbe non-immunogenic in humans. These monoclonal antibodies are secretedby hybridomas constructed by fusing lymphocytes to a human x mouseheteromyeloma.

EP 0 605 442 discloses chimeric antibodies which bind human antigens.These antibodies comprise the whole variable region from an Old Worldmonkey and the constant region of a human or chimpanzee antibody. One ofthe advantages suggested in this reference for these constructs is theability to raise antibodies in Old World monkeys to human antigens whichare less immunogenic in humans compared with antibodies raised in amouse host.

New World primates (infraorder—Platyrrhini) comprise at least 53 speciescommonly divided into two families, the Callithricidae and Cebidae. TheCallithricidae consist of marmosets and tamarins. The Cebidae includesthe squirrel monkey, titi monkey, spider monkey, woolly monkey,capuchin, uakaris, sakis, night or owl monkey and the howler monkey.

Evolutionarily distant primates, such as New World primates, are notonly sufficiently different from humans to allow antibodies againsthuman antigens to be generated, but are sufficiently similar lo humansto have antibodies similar to human antibodies so that the host does notgenerate an anti-antibody immune response when such primate-derivedantibodies are introduced into a human.

Previous studies have characterised the expressed immunoglobulin heavychain repertoire of the Callithrix jacchus marmoset (von Budingen etal., 2001, Immunogenetics; 53:557-563). Six IGHV subgroups wereidentified which showed a high degree of sequence similarity to theirhuman IGHV counterparts. The framework regions were more conserved whencompared to the complementarity determining regions (CDRs). The degreeof similarity between C. jacchus and human 1GHV sequences was less thanbetween non-human Old World primates and humans.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides a chimeric antibodypolypeptide comprising an antigen binding site, wherein the antigenbinding site comprises a human variable domain having at least one NewWorld Primate CDR.

In a second aspect the present invention provides a method of producingan antibody polypeptide according to the first aspect of the invention,the method comprising the steps of:

-   -   (i) providing an acceptor sequence encoding a human variable        domain; and    -   (ii) replacing a CDR sequence of the variable domain with a        donor CDR sequence, wherein the donor sequence is a New World        Primate CDR sequence.

In a third aspect the present invention provides a chimeric domainantibody (dAb) which binds human TNF-α, the dAb comprising animmunoglobulin heavy or light chain variable domain, wherein saidvariable domain comprises at least one New World Primate CDR.

In a fourth aspect the present invention provides a pharmaceuticalcomposition comprising an effective amount of an antibody polypeptideaccording to the first or third aspects of the invention, together witha pharmaceutically acceptable carrier or diluent.

BRIEF DESCRIPTION OF THE FIGSURES

FIG. 1 shows the amino acid (SEQ ID No:6) and nucleotide sequence (SEQID No:5) of the acceptor dAb.

FIG. 2 shows the nucleotide and amino acid sequences of eleven (11)marmoset and six. (6) Owl monkey Vκ gene segments.

FIG. 3 shows the acceptor dAb amino acid and nucleotide sequence (bothstrands). The restriction digest sites for Kpn I and San DI whichexcises a region including the CDR2 is indicated in the figure. CDR2residues removed are indicated in underline.

FIG. 4 shows sequence alignments showing oligonucleotides used duringcloning and final sequence confirmation of the nucleotide (A) and aminoacid (B) sequences shown in FIG. 2.

FIG. 5 demonstrates the ability of CDR2-grafted dAbs to inhibit thebinding of TNF to recombinant TNF receptor. The dAbs tested were asfollows: Owl Monkey 1 (CDR=YAATKLQS; SEQ ID No:1), Owl Monkey 2(CDR=YEASSLQS; SEQ ID No:2), Marmoset 1 (CDR=YEASKLQS; SEQ ID No:3),Marmoset 2 (CDR=YSASNLET; SEQ ID No:4) and Acceptor dAb (CDR=YSASELQS;SEQ ID No:49).

FIG. 6 demonstrates the improved ability of Compounds 100 and 123 toneutralise the cytotoxic activity of TNF on mouse L929 fibroblastsrelative to Compound 145.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention provides a chimeric antibodypolypeptide comprising an antigen binding site, wherein the antigenbinding site comprises a human variable domain having at least one NewWorld Primate CDR.

In a third aspect the present invention provides a chimeric domainantibody (dAb) which binds human TNF-α, the dAb comprising animmunoglobulin heavy or light chain variable domain, wherein saidvariable domain comprises at least one New World Primate CDR.

In a fourth aspect the present invention provides a pharmaceuticalcomposition comprising an effective amount of an antibody polypeptideaccording to the first or third aspects of the invention, together witha pharmaceutically acceptable carrier or diluent.

In an embodiment of the present invention the human variable domaincomprises at least one human framework region having an amino acidsequence encoded by a human germline antibody gene segment, or an aminoacid sequence comprising up to 5 amino acid differences relative to theamino acid sequence encoded by the human germline antibody gene segment.

The human variable domain preferably comprises four human frameworkregions, FR1, FR2, FR3 and FR4 having amino acid sequences encoded by ahuman germline antibody gene segment, or the amino acid sequences whichcollectively contain up to 10 ammo acid differences relative to theamino acid sequences encoded by said human germline antibody genesegment.

Preferably the human germline antibody gene segment selected from thegroup consisting of DP47, DP45, DP48 and DPK9.

The New World Primate CDR may be any CDR, however, it is preferred thatthe New World Primate CDR is CDR2.

Alternatively the New World Primate CDR is CDR1 or CDR3.

It is also preferred that the New World Primate CDR sequence is agermline New World Primate CDR sequence.

The antibody polypeptide of the present invention is preferably selectedfrom a dAb, scFv, Fab, (Fab′)₂, Fv, disulphide bonded Fv, IgG, and adiabody.

The antibody polypeptide of the present invention is preferably directedagainst TNF-α.

In another preferred embodiment the human variable domain amino acidsequence comprises a Kpn1 restriction site spaced from a SanD1restriction site, said CDR of the human variable domain being betweenthe restriction sites.

It is also preferred that the New World Primate CDR sequence isobtainable from New World Primate DNA by PCR using primer pair VK1BL(SEQ ID No:11)/VK1BL35a (SEQ ID No:12) or primer pair VK1BL (SEQ IDNo:11/VK1BL35b (SEQ ID No:13).

The present invention also provides a chimeric domain antibody (dAb)which binds to human TNF-α, wherein the dAb is a human dAb that bindshuman TNF-α in which at least one of the CDRs is replaced with thecorresponding CDR from a New World Primate.

The present invention also provides a method of producing an antibodypolypeptide according to the first aspect of the invention, the methodcomprising the steps of:

-   -   (i) providing an acceptor sequence encoding a human variable        domain; and    -   (ii) replacing a CDR sequence of the variable domain with a        donor CDR sequence, wherein the donor sequence is a New World        Primate CDR sequence.

It is preferred that in step (ii) said CDR of said human variable domainis replaced by said donor New World Primate CDR using restrictiondigestion and annealing of an oligonucleotide encoding the donor CDRinto the acceptor sequence.

It is preferred that the method further comprises affinity maturing thevariable domain produced in step (ii).

As used herein the term “New World Primate CDR” refers to a CDR sequenceobtained from a New World Primate. The term encompasses modification of1, 2 or 3 ammo acids within the sequence which may be used to achieveimproved antigen binding characteristics or lower immunogenicity. Theterm does not, however, extend to cover modifications which result inthe New World Primate CDR sequence being identical to a human CDRsequence.

As used herein the term “human framework region” refers to a frameworkregion obtained from a human or a human framework region having an aminoacid sequence encoded by a human germline antibody gene segment, or anamino acid sequence comprising up to 5 amino acid differences relativeto the amino acid sequence encoded by the human germline gene segment.The term also encompasses modification of the amino acid sequence of theframework region in order to obtain improved antigen bindingcharacteristics or lower immunogenicity such as disclosed in U.S. Pat.No. 4,816,567, U.S. Pat. No. 5,585,089 and US 20030039649 thedisclosures of which are incorporated herein by reference in theirentirety. Typically where modifications are made the total number ofresidues changed will be 10 or less collectively over the frameworkregions.

In a preferred embodiment the variable domain comprises four frameworkregions, wherein at least one framework region comprises an amino acidsequence derived from a corresponding framework region encoded by ahuman germline immunoglobulin gene.

In a further preferred embodiment the four framework regions compriseamino acid sequences derived from corresponding framework regionsencoded by human germline immunoglobulin genes.

In yet a further preferred embodiment the human germline immunoglobulingene is selected from the group consisting of DP47, DP45, DP48 and DPK9.

The term “domain” as used herein is meant a folded protein structurewhich retains its tertiary structure independently of the rest of theprotein. Generally, domains are responsible for discrete functionalproperties of proteins, and in many cases may be added, removed ortransferred to other proteins without loss of function of the remainderof the protein and/or of the domain.

The term immunoglobulin or antibody “variable domain” as used herein isa term of art, and includes a folded polypeptide domain comprisingsequences characteristic of immunoglobulin or antibody heavy or lightchain variable domains and which specifically binds an antigen.

The term “immunoglobulin” as used herein refers to a family ofpolypeptides which retain the immunoglobulin fold characteristic ofantibody molecules, which contains two β sheets and, usually, aconserved disulphide bond. Members of the immunoglobulin superfamily areinvolved in many aspects of cellular and non-cellular interactions invivo, including widespread roles in the immune system (for example,antibodies, T-cell receptor molecules and the like), involvement in celladhesion (for example the ICAM molecules) and intracellular signalling(for example, receptor molecules, such as the PDGF receptor). Thepresent invention is applicable to all immunoglobulin superfamilymolecules which possess binding domains. Preferably, the presentinvention relates to antibody polypeptides.

New World primates (infraorder—Platyrrhini) comprise at least 53 speciescommonly divided into two families, the Callithricidae and Cebidae. TheCallithricidae consist of marmosets and tamarins. The Cebidae includesthe squirrel monkey, titi monkey, spider monkey, woolly monkey,capuchin, uakaris, sakis, night or owl monkey and the howler monkey.

Evolutionarily distant primates, such as New World primates, are notonly sufficiently different from humans to allow antibodies againsthuman antigens to be generated, but are sufficiently similar to humansto have antibodies similar to human antibodies so that the host does notgenerate an anti-antibody immune response when such primate-derivedantibodies are introduced into a human.

Previous studies have characterised the expressed immunoglobulin heavychain repertoire of the Callithrix jacchus marmoset (von Budingen H-C etal., 2001, Immunogenetics; 53:557-563). Six IGHV subgroups wereidentified which showed a high degree of sequence similarity to theirhuman IGHV counterparts. The framework regions were more conserved whencompared to the complementarity determining regions (CDRs). The degreeof similarity between C. jacchus and human IGHV sequences was less thanbetween non-human Old World primates and humans.

In certain embodiments of the present invention the New World primateCDR is from the family Callithricidae.

In yet a further embodiment of the present invention the New Worldprimate CDR is selected from the group consisting of marmosets,tamarins, squirrel monkey, titi monkey, spider monkey, woolly monkey,capuchin, uakaris, sakis, night or owl monkey and the howler monkey.More preferably, the New World primate is a marmoset.

In yet a further embodiment of the present invention the at least oneNew World primate CDR is substantially identical to a CDR encoded by aNew World primate germline immunoglobulin gene.

The term “antibody” as used herein, is intended to refer toimmunoglobulin molecules comprised of two heavy chains or immunoglobulinmolecules comprised of four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds. Each heavychain is comprised of a heavy chain variable region (HCVR or V_(H)) anda heavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain is comprisedof a light chain variable region (LCVR or V_(L)) and a light chainconstant region. The light chain constant region is comprised of onedomain, C_(L). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDR), interspersed with regions that are more conserved, termedframework regions (PR). Each V_(H) and V_(L) is composed of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term “antibody polypeptide” as used herein refers to a polypeptidecomprising one or more components or derivatives of an immunoglobulinthat exhibit the ability to bind to an antigen. It has been shown thatthe antigen-binding function of an antibody can be performed byfragments of a full length antibody. Examples of binding fragmentsencompassed within the term “antibody polypeptide” include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and C_(H)1 domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb fragment (Ward et al, 1989, Nature341:544-546) which consists of a single V_(H) domain, or a V_(L) domain(van den Beucken et al, 2001, J. Mol. Biol, 310, 591-601); and (vi) anisolated complementarity determining region (CDR). Furthermore, althoughthe two domains of the Fv fragment, V_(L) and V_(H), are coded byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the V_(L) and V_(H) regions pair to form monovalent molecules(known as single chain Fv (scFv); (see eg Bird et al., 1988, Science242:423-426 and Huston et al., 1988 Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain Fvs are also intended to be encompassedwithin the term “antigen-binding portion” of an antibody. Other forms ofsingle chain Fvs and related molecules such as diabodies or triabodiesare also encompassed. Diabodies are bivalent antibodies in which V_(H)and V_(L) domains are expressed on a single polypeptide chain, but usinga linker that is too short to allow for pairing between the two domainson the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, et al., 1993, Proc. Natl. Acad. Sci. USA,90:6444-6448; Poljak, et al., 1994, Structure, 2:1121-1123).

Thus in certain embodiments of the present invention the antibodypolypeptide is selected from the group consisting of a dAb, scFv, Fab,F(ab′)₂, Fv, disulphide bonded Fv, a diabody and IgG.

Preferably, the antibody polypeptide further comprises a human ornon-human primate constant region sequence. Examples of non-humanprimates include, but are not limited to, chimpanzees, oranguatangs andbaboons.

The constant region sequence (Fc portion) is preferably obtained from ahuman or non-human primate immunoglobulin sequence. The primate sequencemay be a New World primate or an Old World primate sequence. SuitableOld World primates include chimpanzee, or other hominid ape eg. gorillaor orangutan, which because of their close phylogenetic proximity tohumans, share a high degree of homology with the human constant regionsequence. Sequences which encode for human or primate constant regionsare available from databases including e.g. The National Centre forBiotechnology Information protein and nucleotide databases, The KabatDatabase of Sequences of Proteins of Immunological Interest.

In a preferred embodiment of the present invention the antibodypolypeptide is a domain antibody (dAb).

Domain antibodies (dAb) are small functioning binding units ofantibodies and correspond to the variable regions of either the heavy(V_(H)) or light (V_(L)) chains of antibodies. Domain antibodies have amolecular weight of approximately 13 kDa, or less than one tenth thesize of a full antibody.

Antibody light chains are referred to as either kappa or lambda lightchains and the heavy chains as gamma, mu, delta, alpha or epsilon. Thevariable region gives the antibody its specificity. Within each variableregion are regions of hypervariability, otherwise known ascomplementarity determining regions (CDRs) which are flanked by moreconserved regions referred to as framework regions. Within each variableregion are three CDRs and four framework regions.

In contrast to conventional antibodies, domain antibodies are wellexpressed in bacterial, yeast and mammalian systems. Their small sizeallows for higher molar quantities per gram or product, thus providing asignificant increase in potency per dose. In addition, domain antibodiescan be used as a building block to create therapeutic products such asmultiple targeting dAbs in which a construct containing two or morevariable domains bind to two or more therapeutic targets, or dAbstargeted for pulmonary or oral administration.

An increase in binding is demonstrated by a decrease in K_(D)(k_(off)/k_(on)) for the antibody or antigen binding portion thereof. Anincrease in potency is demonstrated in biological assays. For example,assays that can be used to measure the potency of the antibody orantigen-binding portion thereof include the TNFα-induced L929cytotoxicity neutralisation assay, IL-12-induced human PHA-activatedperipheral blood mononuclear cell (PBMC) proliferation assay, and RANKLmediated osteoclast differentiation of mouse splenocytes (Stem, 1990,Proc. Natl. Acad. Sci. USA 87:6808-6812; Kong, et al., 1990, Nature397:315-323; Matthews and Neale in Lymphokines and Interferons, aPractical Approach, 1987, M. J. Clemens, A. G. Morris and A. J. H.Gearing, eds., IRL Press, p. 221).

The CDR sequences may be obtained from several sources, for example,databases e.g. The National Centre for Biotechnology Information proteinand nucleotide databases www.ncbi.nlm.nih.gov, The Kabat Database ofSequences of Proteins of Immunological Interest www.kabatdatabase.com,or the IMGT database www.imgt.cines.fr. Alternatively, the CDR regionscan be predicted from the V_(H) and V_(L) domain repertoire (see forexample Kabat and Wu, 1971, Ann. NY Acad. Sci. 190:382-393). The CDRsequence may be a genomic DNA or a cDNA.

There are a number of ways in which a replacement CDR may be graftedinto a variable domain sequence and such methods will be familiar tothose skilled in the art. The preferred method of the present inventioninvolves replacement of the CDR2 in the variable region domain viaprimer directed mutagenesis. This method consists of annealing asynthetic oligonucleotide encoding a desired mutations to a targetregion where it serves as a primer for initiation of DNA synthesis invitro, extending the oligonucleotide by a DNA polymerase to generate adouble-stranded DNA that carries the desired mutations, and ligating andcloning the sequence into an appropriate expression vector.

In one embodiment of the invention, the variable domain sequence intowhich the CDR is grafted is the “dAb acceptor sequence” (designatedCompound 128; SEQ ID No:6) provided in FIG. 1.

As used herein the term “chimeric” is meant that the antibodypolypeptide or domain antibody includes sequences from more than onespecies.

The anti-human TNF-α dAb according to the invention can be used todetect human TNF-α for example in a biological sample, such as serum orplasma using a conventional immunoassay, such as an enzyme linkedimmunosorbent assay (ELISA), a radioimmunoassay (RIA) or tissueimmunohistochemistry. The anti-human TNF-α dAb according to theinvention can be assayed in biological fluids by a competitionimmunoassay using recombinant human TNF-α standards label led with adetectable substance and an unlabelled anti-human TNF-α antibody.

The anti-human TNF-α dAb according to the invention may also be used todetect TNF-α from species other than humans eg. chimpanzee, marmoset,rhesus, mouse, pig.

The anti-human TNF-α dAb according to the invention may also be used incell culture applications where it is desired to inhibit TNF-α activity.

The invention also provides a method for treating a disordercharacterised by human TNF-α activity in a human subject, comprisingadministering to the subject a pharmaceutical composition according tothe second aspect of the invention.

A disorder characterised by human TNF-α activity is intended to includediseases and other disorders in which the presence of TNF-α in a subjectsuffering from the disorder has been shown to be or is suspected ofbeing either responsible for the pathophysiology of the disorder or afactor which contributes to a worsening of the disorder. Preferably, thedisorder characterised by human TNF-α activity is selected from thegroup consisting of inflammation, inflammatory diseases, sepsis,including septic shock, endotoxic shock, gram negative sepsis and toxicshock syndrome; autoimmune disease, including rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergy,multiple sclerosis, autoimmune diabetes, autoimmune uveitis andnephrotic syndrome; infectious disease, including fever and myalgias dueto infection and cachexia secondary to infection; graft versus hostdisease; tumour growth or metastasis; pulmonary disorders includingadult respiratory distress syndrome, shock lung, chronic pulmonaryinflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis andsilicosis; inflammatory bowel disorders including Crohn's disease andulcerative colitis; cardiac disorders; inflammatory bone disorders,hepatitis, coagulation disturbances, burns, reperfusion injury, keloidformation and scar tissue formation.

In a fourth aspect, the invention provides a pharmaceutical compositioncomprising an effective amount of the antibody polypeptide according tothe first aspect of the invention or a chimeric domain antibodyaccording to the third aspect of the invention, together with apharmaceutically acceptable carrier or diluent.

A “pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal-agents,isotonic and absorption delaying agents, and the like which arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol, and the like as well ascombinations thereof. In many cases it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable substances such as welling or minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers.

The composition may be in a variety of forms, including liquid,semi-solid and solid dosage forms, such as liquid solutions (eginjectable and infusible solutions), dispersions or suspensions,tablets, pills, powders, liposomes and suppositories. Preferably, thecomposition is in the form of an injectable solution for immunization.The administration may be intravenous, subcutaneous, intraperitoneal,intramuscular, transdermal, intrathecal, and intra-arterial.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The compositions can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high drug concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e. antibody polypeptide) into the required amount in anappropriate solvent with one or a combination of ingredients listedabove, followed by filtered sterilisation.

The composition may also be formulated as a sterile powder for thepreparation of sterile injectable solutions. The proper fluidity of asolution can be maintained by for example, use of a coating such aslecithin and/or surfactants.

In certain embodiments, the active compound may be prepared with acarrier that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems.

Compatible polymers may be used such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters andpolylactic acid.

The composition may also be formulated for oral administration. In thisembodiment, the antibody polypeptide may be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet.

The composition may also be formulated for rectal administration.

Supplementary active compounds can also be incorporated into thecomposition. The antibody polypeptide may be co-formulated with and/orco-administered with one or more additional therapeutic agents eg.anti-inflammatory compounds, soluble TNF-α receptor or a chemical agentthat inhibits human TNF-α production, or antibodies that bind othertargets such as cytokines or cell surface molecules. Alternatively, itmay be co-administered with a soluble immunochemical reagent such asprotein A, C, G or L.

An effective amount may include a therapeutically effective amount orprophylactically effective amount of the antibody polypeptide of theinvention. A therapeutically effective amount refers to an amounteffective at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A prophylactically effective amount refersto an amount effective, at dosages and for periods of lime necessary, toachieve the desired prophylactic result.

In a preferred embodiment the composition is administered to mammals,preferably humans or primates.

In order that the nature of the present invention may be more clearlyunderstood, preferred forms thereof will now be described with referenceto the following non-limiting examples.

EXAMPLE 1

Materials and Methods

Isolation of New World Primate VL Genes

Marmoset (genus Callidirix, species unknown) and Owl monkey (Aotustrivirgatus) genomic DNA were obtained from the European Collection ofCell Cultures (ECACC), catalogue numbers 85011419 and 90110510respectively. Marmoset DNA was derived from cell line B95-8 while Owlmonkey DNA came from cell line OMK 637-69.

Degenerate primers based on human Vκ leader sequences and recombinationsignal sequences (RSS) were derived from Walter and Tomlinson, AntibodyEngineering: A Practical Approach (1996). The primers used foramplification of germline Vκ DNA were as follows:

Primer VK1BL AATCKCAGGTKCCAGATG (SEQ ID No: 11) Primer VK1BL35aGTTYRGGTKKGTAACACT (SEQ ID No: 12) Primer VK1BL35b ATGMCTTGTWACACTGTG(SEQ ID No: 13)

Genomic PCR (30 cycles) was performed using Taq polymerase with eitherprimer pair VK1BLxVK1BL35a or VK1LxVK1BL35b. There was overlap betweenthe sequences cloned and the two primer sets used.

PCR products were cloned into Invitrogen's TOPO TA cloning kit (Cat NoK4500-01) and sequenced with M13 forward and pUC reverse primers.Sequence was confirmed in forward and reverse directions. In order tofurther confirm key sequences were not subject, to PCR errors, the PCRand cloning process was repeated twice for marmoset sequences.Nucleotide (SEQ ID Nos:14-24 and SEQ ID Nos:36-41) and amino acid (SEQID Nos:25-35 and SEQ ID Nos:42-47) sequences are given in FIG. 2.Marmoset sequences 1, 2 and 3 were confirmed. Sequences 4, 5, 6, 7 and 8were seen only in the initial PCR. Sequences 9, 10 and 11 were seen onlyin the repeat (i.e. second) PCR and cloning.

Oligo Synthesis and Cloning into Acceptor Sequence

Four CDR sequences, namely YAATKLQS (SEQ ID No:1) from Owl monkeysequence 1 (SEQ ID No:42), YEASSLQS (SEQ ID No:2) from Owl monkeysequence 2 (SEQ ID No:43), YEASKLQS (SEQ ID No:3) from Marmoset sequence1 (SEQ ID No:25), and YSASNLET (SEQ ID No:4) from Marmoset sequence 2(SEQ ID No:26), were chosen from the amino acid sequences shown in FIG.2 as indicated. Owl Monkey sequence 5, YYASSLQS (SEQ ID No:48) was foundto be identical to GI6176295 an Aotus nancymaae (Ma's night monkey) cDNAsequence, all other sequences were unique.

An acceptor variable region (anti-TNF domain antibody) sequence in theexpression vector (Domantis proprietary vector) was digested (25 μg)sequentially with KpnI and SanDI which excises the majority of FR2 aswell as CDR2 as indicated on the restriction digest map. The vector wasthen gel purified to remove the excised wild-type FR2 and CDR2 sequence.

Oligo annealing was performed by incubating oligo pairs (500 pmol ofeach as shown in FIGS. 4A and 4B) at 95° C. for 5 minutes followed by65° C. for 5 minutes and then allowed to reach room temperature slowlyon a hot block. Overlaps were then filled in during a Klenow reaction inthe presence of dNTPs.

Affinity Maturation

The marmoset CDR-grafted dAb Compound 145 (SEQ ID No:7) was affinitymatured by constructing 14 separate libraries, each a diversification ofthe sequence of SEQ ID No:7 at a single amino acid residue. The selectedresidues are shown shaded below.

The selection was based upon residues in CDR1 and CDR3 that are known tobe diversified in the mature human Ig repertoire, and framework residuesthat have been observed to produce functional proteins after mutagenesisin related dAbs. For each of the selected residues, complimentaryforward and reverse PCR primer pairs were designed with NKK degeneracy,and two initial PCR reactions were performed each with a singlemutagenic primer and flanking primer. After clean-up, the two PCRproducts were annealed and then amplified using flanking primers alone(splicing by overlap extension of PCR; Lowman H. L. & Clackson T. (eds),Phage Display: A practical approach, Oxford University Press , Oxford,UK). Clones were initially screened by ELISA using solid-phase TNF, andpositive clones were sequenced. dAb protein was purified from the bestclones and evaluated for potency in receptor binding assays and L929cytotoxicity assays. Compounds 100 (SEQ ID No:9) and 123 (SEQ ID No:8)were found to have improved TNF-neutralization relative to the parentdAb, Compound 145 (SEQ ID No:7).

Combination of the affinity-enhancing substitutions of Compounds 100(SEQ ID No:9) and 123 (SEQ ID No:8), yielded an anti-TNF dAb withfurther improved potency in the L929 cytotoxicity assay (Compound 196;SEQ ID No:10).

Results

Potency of Anti-TNF dAb Clones in Receptor Binding Assay (RBA) andCytotoxocity Assay

The ability of the anti-TNF dAbs to inhibit TNF binding to its receptorand to neutralize TNF-mediated cytotoxicity of L929 cells was conductedas follows:

Receptor Binding Assay

dAbs diversified in the 14 selected positions were tested for theability to inhibit the binding of TNF to recombinant TNF receptor 1(p55). Briefly, Maxisorp plates were incubated overnight with 30 mg/mlanti-human Fc mouse monoclonal antibody (Zymed, San Francisco, USA). Thewells were washed with phosphate buffered saline (PBS) containing 0.05%Tween-20 and then blocked with 1% BSA in PBS before being incubated with100 ng/ml TNF receptor 1 Fc fusion protein (R&D Systems, Minneapolis,USA). Each dAb was mixed with TNF which was added to the washed wells ata final concentration of 10 ng/ml. TNF binding was detected with 0.2mg/ml biotinylated anti-TNF antibody (HyCult biotechnology, Uben,Netherlands) followed by 1 in 500 dilution of horse radish peroxidaselabelled streptavidin (Amersham Biosciences, UK) and then incubationwith TMB substrate (KPL, Gaithersburg, USA). The reaction was stopped bythe addition of HCl and the absorbance was read at 450 nm. Anti-TNF dAbactivity lead to a decrease in TNF binding and therefore a decrease inabsorbance compared with the TNF only control (FIG. 5).

L929 Cytotoxicity Assay

Anti-TNF dAbs identified by the minilibrary diversification approach,including Compounds 100 (SEQ ID No:9) and 123 (SEQ ID No:8), were alsotested for the ability to neutralise the cytotoxic activity of TNF onmouse L929 fibroblasts (Evans, T., 2000, Molecular Biotechnology 15,243-248). Briefly, L929 cells plated in microtitre plates were incubatedovernight, with anti-TNF dAb, 100 pg/ml TNF and 1 mg/ml actinomycin D(Sigma, Poole, UK). Cell viability was measured by reading absorbance at490 nm following an incubation with[3-(4,5-dimethylthiazol-2-yl)-5-(3-carbboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(Promega, Madison, USA). Anti-TNF dAb activity lead to a decrease in TNFcytotoxicity and therefore an increase in absorbance compared with theTNF only control. The results, in comparison with the parent dAbCompound 145 (SEQ ID No:7) are presented in FIG. 6.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated clement, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

All publications mentioned in this specification are herein incorporatedby reference. Any discussion of documents, acts, materials, devices,articles or the like which has been included in the presentspecification is solely for the purpose of providing a context for thepresent invention. It is not to be taken as an admission that any or allof these matters form part of the prior art base or were common generalknowledge in the field relevant to the present invention as it existedin Australia or elsewhere before the priority date of each claim of thisapplication.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, lobe considered in all respects as illustrative and notrestrictive.

1. A chimeric antibody polypeptide comprising an antigen binding site,wherein the antigen binding site comprises a human variable domainhaving at least one New World Primate CDR.
 2. The antibody polypeptideof claim 1, wherein the human variable domain comprises at least onehuman framework region having an amino acid sequence encoded by a humangermline antibody gene segment, or an amino acid sequence comprising upto 5 amino acid differences relative to the amino acid sequence encodedby a human germline antibody gene segment.
 3. The antibody polypeptideof claim 2, wherein the human variable domain comprises four humanframework regions, FR1, FR2, FR3 and FR4 having amino acid sequencesencoded by a human germline antibody gene segment, or the amino acidsequences of FR1, FR2, FR3 and FR4 collectively containing up to 10 ammoacid differences relative to the amino acid sequences by said humangermline antibody gene segment.
 4. The antibody polypeptide according toclaim 2, wherein the framework regions are encoded by a human germlineantibody gene segment selected from the group consisting of DP47, DP45,DP48 and DPK9.
 5. The antibody polypeptide of claim 1, wherein said NewWorld Primate CDR is CDR2.
 6. The antibody polypeptide of claim 1,wherein said New World Primate CDR is CDR1 or CDR3.
 7. The antibodypolypeptide of claim 1, wherein said New World Primate CDR sequence is agermline New World Primate CDR sequence.
 8. The antibody polypeptide ofclaim 1, wherein the antibody polypeptide is selected from the groupconsisting of a dAb, scFv, Fab, (Fab′)₂, Fv, disulphide bonded Fv, IgG,and a diabody.
 9. The antibody polypeptide of claim 1, wherein theantigen is TNF-α.
 10. The antibody polypeptide of claim 1, wherein theNew World Primate is a Callithricidae.
 11. The antibody polypeptide ofclaim 10, wherein the New World Primate is a marmoset.
 12. The antibodypolypeptide of claim 1, wherein the human variable domain amino acidsequence comprises a Kpn1 restriction site spaced from a SanD1restriction site, said CDR of the human variable domain being betweenthe restriction sites.
 13. The antibody polypeptide of claim 1, whereinsaid New World Primate CDR sequence is obtainable from New World PrimateDNA by PCR using primer pair VK1BL (SBQ ID No:11)/VK1BL35a (SEQ IDNo:12) or primer pair VK1BL (SEQ ID No:11)/VK1BL35b (SEQ ID No:13). 14.A chimeric domain antibody (dAb) which binds to human TNF-α, wherein thedAb is a human dAb that binds human TNF-α in which at least one of theCDRs is replaced with the corresponding CDR from a New World Primate.15. A chimeric dAb according to claim 14 wherein the replaced CDR isCDR2.
 16. A chimeric dAb according to claim 14 wherein the New WorldPrimate is a marmoset.
 17. A method of producing an antibody polypeptideas defined in claim 1, the method comprising (i) Providing an acceptorsequence encoding a human variable domain; and (ii) Replacing a CDRsequence of the variable domain with a donor CDR sequence, wherein thedonor CDR sequence is a New World Primate CDR.
 18. The method of claim17, wherein in step (ii) said CDR of said human variable domain isreplaced by said donor New World Primate CDR using restriction digestionand annealing of an oligonucleotide encoding the donor CDR into theacceptor sequence.
 19. The method of claim 17, further comprising (iii)affinity maturing the variable domain produced in step (ii).
 20. Amethod of producing a chimeric dAb as defined in claim 14, the methodcomprising (i) Providing an acceptor sequence encoding a human variabledomain; and (ii) Replacing a CDR sequence of the variable domain with adonor CDR sequence, wherein the donor CDR sequence is a New WorldPrimate CDR.
 21. The method of claim 20, wherein in step (ii) said CDRof said human variable domain is replaced by said donor New WorldPrimate CDR using restriction digestion and annealing of anoligonucleotide encoding the donor CDR into the acceptor sequence. 22.The method of claim 20, further comprising (iii) affinity maturing thevariable domain produced in step (ii).